GTP-dependent regulatory proteins (G proteins) modulate numerous intracellular processes including many that are regulated by extracellular stimuli (such as hormones, light, odorants, etc.). For example, the heterotrimeric G proteins mediate hormonal regulation of effector molecules such as adenylyl cyclase or phospholipase C (PLC) to alter production of the second messengers, cAMP or inositol 1,4,5-trisphosphate and diacylglycerol, respectively. Monomeric G proteins such as the Ras, Rho, or Rab families mediate effects of growth factors or act more directly in regulating cell shape or intracellular vesicle traffic, respectively. The long term goal of this research has been and is the elucidation of pathways and mechanisms for regulation through the G proteins. Among the heterotrimeric G proteins, the ubiquitously expressed subfamily consisting of G12 and G13 stands out as the least characterized. Progress in this group has resulted in the purification of G13 and identification of its role in the regulation of Ca2+ channels. Nevertheless, the immediate downstream effector of G13 is unknown. One goal of this application is to further characterize the properties of purified G13 and its interaction with receptors. Examination of the function of G13 will include a complementary combination of pbenotypic characterization of cells overexpressing the protein and biochemical approaches to isolate and characterize effector proteins regulated by G13. Many hormones which stimulate the activity of PLC also increase the activity of phospholipase D (PLD) and thus, the production of phosphatidic acid (PA), a putative second messenger and precursor to diacylglycerol and lysophosphatidic acid (LPA). Recent progress demonstrated that PLD activity could be regulated by a small monomeric G protein, ARF. This application proposes to examine several aspects of PLD regulation. Aims include purification, cloning, and expression of the ARF-sensitive PLD (ARF-PLD) enzyme, and characterization of the crude and purified enzyme with respect to structure, catalytic activity, and regulation by ARF and other potential factors. Further pursuits include attempts to characterize hormone regulated PLD activities which could include identification of novel enzymes directly regulated by heterotrimeric G proteins or determining mechanism by which receptors stimulate ARF/PLD. Evidence has mounted for a prominent role of ARF in intracellular protein trafficking. Therefore, experiments are also proposed to determine the potential role of ARF-PLD and potential changes in membrane lipids in the mechanisms for vesicle traffic. Progress from these experiments will contribute to further delineation of G-protein mechanisms and thus our understanding of regulation by hormones. The study of PLD regulation by ARF may further elucidate fundamental mechanisms for intracellular transport and secretion of proteins.